Pili or fimbriae are present on the surface of many gram-positive pathogens, including Corynebacterium diphtheriae, enterococci and streptococci. These fiber-like protein polymers play a pivotal role in many facets of bacterial pathogenesis such as adherence to host tissues, invasion, biofilm formation and modulation of host immunity. The assembly of these pili on the bacterial cell wall occurs by a sortase-dependent mechanism, which was first demonstrated in C. diphtheriae, the causative agent of diphtheria. C. diphtheriae expresses three distinct heterotrimeric pilus structures, named SpaA-, SpaD-, and SpaH-type pili according to the major subunit that forms the pilus shaft. Well characterized is the SpaA-type pilus that is encoded by the spaA-srtA- spaB-spaC locus. This archetype pilus is composed of SpaA forming the shaft, SpaC located at the tip and SpaB at the pilus base. Importantly, the SpaA pili mediate corynebacterial adherence to pharyngeal epithelial cells, the major site of C. diphtheriae infection. We showed that polymerization of the pilus subunits requires a cognate transpeptidase enzyme named pilin-specific sortase (SrtA) via lysine-mediated transpeptidation. The resulting polymer is joined to the cell wall by the housekeeping sortase SrtF. Recently, a great deal of progress has been made on the biology of Gram-positive pili, establishing the prominent roles of pili in pathogenesis as well as their suitability for developing multivalent vaccines against streptococcal pathogens. However, there are major gaps in our understanding of (a) how surface expression of pili is controlled, (b) how pilins are assembled in a specific order, and (c) what governs the decision between pilus polymerization and cell wall anchoring and its ramification in bacterial pathogenesis. In the current proposal, we seek answers to these fundamental questions by using a multidisciplinary approach. Specifically, we seek to delineate the mechanism that controls surface assembly of pili by characterizing SrtA-SrtF interactions with their substrates and SarA, a novel membrane bound protein required for SrtF-mediated cell wall anchoring, and by discovering specific trans-acting factors that regulate srtF expression. Secondly, we aim to further elucidate the fundamental mechanisms of pilus biogenesis by deciphering the molecular determinants and the underlying interactions that orchestrate the ordered assembly of the pilus and by uncovering potential chaperones and cofactors involved in pilus biogenesis. Finally, we aim to establish two paradigmatic principles that surface assembly of corynebacterial pili significantly contributes to bacterial pathogenesis and that a vaccine combining pilus and toxin antigens enhances protective efficacy. The results generated will certainly provide not only a clear understanding of the basic mechanisms of pilus assembly and pilus-mediated pathogenesis in Gram-positive bacteria but also knowledge that improves health.